Our objective is to develop and validate cellular and molecular imaging techniques that can be used to track recruitment and differentiation of progenitor cells into cardiomyocytes in vivo. In preliminary studies, we showed that adult peripheral blood CD34+ cells injected intraventricularly into severe combined immune- deficient (SCID) mice could transform into myocytes and smooth muscle cells in the heart. However, it is difficult to follow the recruitment of progenitor cells into the heart and their subsequent differentiation/fusion into various cardiac cells in vivo. In this proposal, we wish to develop cellular and molecular imaging techniques that will allow us to reliably track recruitment of progenitor cells into the injured heart and to follow the time course of their differentiation or fusion into cardiomyocytes. Human stem cells derived from different sources will be assayed in a SCID mouse myocardial regeneration model for selection of the best source of stem cells;the selected cells then will be genetically modified using retroviral or lentiviral vectors bearing a dual positron emission tomography (PET) reporter gene construct. This construct will encode for a constitutively expressed "beacon" reporter gene-the truncated human thymidine kinase type two fused with red fluorescent protein (hTK2RFP)-and a "sensor" reporter gene, consisting of the herpes virus type one thymidine kinase fused with green fluorescent protein (sr39TKGFP) controlled by the a-myosin heavy chain promoter. In vivo PET imaging of the hTK2RFP "beacon" reporter will be accomplished with hTK2-specific radiotracer [18FJFEAU. PET imaging of the sr39TKGFP "sensor" will be performed with sr39HSV1-TK specific radiotracer [18F]FHBG. Importantly, PET imaging with [18F]FHBG does not detect the hTK2RFP "beacon" reporter expression. Repetitive whole body in vivo imaging of "beacon" gene expression will provide quantitative information about the dynamics and patterns of "reporter cell" migration, engraftment, and viability of progeny cells. Most importantly, PET images of "sensor" gene expression will reflect the status of cardiomyocyte-specific differentiation of transplanted progenitors and the distribution of progeny cells. The PET images will be co-registered with the magnetic resonance (MR) images, which will be obtained in the same animals for better anatomic localization of PET-detected stem cells and their progeny tissues. This approach will provide insights into the fate of human stem cell transplantation into the heart.